Globally, cancer is one of humanity's most pressing conditions, with over 14.1 million new cases and 8.2 million deaths each year [1]. Tumor metastasis occurs when tumors shed cancer cells into the bloodstream and lymphatic system [2]. The tumor cells persisting in the peripheral blood stream are termed as circulating tumor cells (CTCs) [3] and can be detected by liquid biopsy (blood withdrawal) [4]. CTC counts demonstrate correlation with patient survival [5, 6] and cancer progression [7].
In the management of biological diseases, early detection and intervention is the key to promote therapeutic success. For leukemia, the golden standard is bone marrow biopsy [8], which is undesirable for several reasons; there are issues associated with high costs, the complexity of surgical procedure, the discomfort of such an invasive procedure as well as an increased risk of mortality. Due to these factors, monitoring mutations or blast cell levels from bone marrow biopsies is a tedious process as these procedures have to be carried out on a routine basis. In cases where these procedures cannot be done (e.g. patient is too weak for surgery), the lack of conclusive screens may affect the evaluation of disease and treatment outcome. Clinicians are keen to introduce rapid and efficient screens for leukemia by employing the use of microfluidic-based assays, which is operated with minimal reagents and samples [9].
In patients with acute myeloid leukemia (AML) and other leukemia types, leukemia spreads when immature white blood cells, termed as blast cells, are released from the bone marrow into the circulation. Blast cell counts from bone marrow can be isolated by flow cytometry [10], and the counts act as a diagnostic marker for leukemia. However, the technical limitations of flow cytometry prevent the isolation of blast cells from blood of patients with low blast cell counts (e.g. those with minimal residual disease (MRD). More specific detection methods (MRD<10−5), such as allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) and deep sequencing could be used on bone marrow samples, but these techniques involve a high level of technical complexity and are not applicable to most patients [11]. A “liquid biopsy” approach capitalizing on blood-derived blast cells for leukemia would be a low-cost, less invasive alternative to bone marrow biopsy. The sensitive detection of residual blood-derived blast cells will provide clinicians with therapeutic guidelines and bring tremendous benefits in the monitoring of patient prognosis [12].
At present, there are no procedures to enrich blast cells from blood. Comparisons of more sensitive detection methods, such as deep sequencing, has been used to demonstrate the ineffectiveness of flow cytometry on detecting bone marrow blast cells. If applied to blood-derived blast cells, flow cytometry will lead to false negatives as the proportion of blast cells in blood is relatively lower than that in bone marrow, and diluted amongst other blood cell populations (>5%). Patients with minimal residual disease (MRD) have 1 cancer cell in 10,000 or 100,000 leukocytes, while those with chronic stages of leukemia present even lower levels of blast cells (<5%). Low residual disease levels (MRD<10-5) are often not detectable by existing diagnostic procedures.
We previously demonstrated the use of inertial-based microfluidics for sorting circulating tumor cells from peripheral blood of patients with solid tumors [13, 14]. We also demonstrated the enrichment of infected malaria blood cells with relevance in disease detection using inertial-based microfluidics [15]. In contrast to other cell sorting microfluidics [16-18], inertial-based microfluidics allows high processing rates. Cell sorting microfluidics are hindered by two common limitations: 1) generating large output volumes due to the need of high dilution factors and; 2) slow processing speed due to compact cellular interactions which leads to biofouling (clogging) of the device. Subsequent steps to concentrate outputs lead to high degrees of target cell losses, further compromising the sensitivity of target cell detection.
Conventional macroscale methods for separation of cells include physical filtration using a membrane-based filter and density gradient centrifugation which exploit differences in cell size, deformability, and density to filter out target cells. These techniques are labor-intensive and require multi-step sample preparations which may introduce artifacts or lead to loss of desired cells. Membrane filtration methods are also easily susceptible to clogging and require frequent cleaning. Further, evidence of mechanical stress-induced changes in original phenotype of target cells subjected to filtration and centrifugation techniques has also been reported. Recently, inertial microfluidic devices were explored as a filter-less size-based cell fractionation method [19][20].
However, there is a continuing need to develop simpler and more efficient techniques to process blood samples that can minimize cell loss and maintain the original target cell phenotype for subsequent analysis.